Capacitation dependent changes in the sperm plasma membrane influence porcine gamete interaction

Although the sperm cell was first seen through Van Leeuwenhoek’s microscope in the late seventieth century and despite much effort in the last 40 years in particular, we still do not know a great deal of the sperm cell and its interaction with the oocyte. Mammalian sperm-oocyte interaction is a complex process requiring the activation of the sperm cell (called capacitation), recognition and binding between the oocyte and the sperm cell, induction of the sperm acrosome reaction (excretion of enzymes from the acrosome i.e. specialised large vesicle in the sperm head), and subsequently penetration of the oocyte’s extracellular matrix (the zona pellucida) followed by fusion of the two gametes. The plasma membrane of the sperm head is pivotal in all of these processes. In order to understand more of the biochemical nature of sperm-oocyte interactions, we isolated the plasma membrane of the sperm head. Chapter 2 describes the membrane isolation from boar (pig) semen and a new method to characterise these preparations with lectins specific for different sperm membranes. In chapter 3 we investigated the differences in tyrosine phosphorylation status of plasma membrane proteins isolated from fresh, in vitro capacitated and mock-incubated sperm cells. Capacitation of sperm cells by incubation at 38°C with bovine serum albumin, calcium and bicarbonate induced tyrosine phosphorylation of at least 3 plasma membrane proteins. To study the role of these proteins in sperm-oocyte interaction we mimicked this contact by application of the plasma membranes isolated from capacitated sperm cells to an affinity column made of purified zona pellucida fragments. In chapter 4 we show that the binding fraction eluted with 1 M NaCl contained two tyrosine-phosphorylated proteins. These two proteins might be the sperm receptor(s) for the zona pellucida and obtain their binding capacity after tyrosine phosphorylation due to capacitation. In vitro capacitation of sperm cells induces activation-like characteristics (such as increased plasma membrane fluidity) in only a subpopulation of cells. In chapter 5 we determined that sperm cells showing a higher plasma membrane fluidity (merocyanine staining and subsequent sorting in a flow cytometer) upon exposure to bicarbonate have a lower cholesterol content, most likely linked to maturation. Addition of bovine serum albumin (lipid acceptor protein) to these cells resulted in a loss of cholesterol. Using filipin as a cholesterol stain in fluorescence and electron microscopy, we were able to visualise the movement of cholesterol towards the apical site of the sperm head when bicarbonate was present and a removal of cholesterol when both bicarbonate and bovine serum albumin were present. It is very likely that the described capacitation-induced changes in both proteins and lipids in the plasma membrane are linked in order to fertilise the oocyte. The results described in this thesis contribute to our understanding how these tiny highly specialised sperm cells are able to recognise and penetrate the oocyte after activation

Rhodoquinone is synthesized de novo by Fasciola hepatica

Most adult parasitic helminths have an anaerobic energy metabolism in which fumarate is reduced to succinate by fumarate reductase. Rhodoquinone (RQ) is an essential component of the electron transport associated with this fumarate reduction, whereas ubiquinone (UQ) is used in the aerobic energy metabolism of parasites. Not known yet, however, is the RQ and UQ composition during the entire life cycle nor the origin of RQ in parasitic helminths. This report demonstrates the essential function of RQ in anaerobic energy metabolism during the entire life cycle ofFasciola hepatica , as the amount of RQ present reflected the importance of fumarate reduction in various stages. We also studied the origin of RQ, as earlier studies on the protozoan Euglena gracilis suggested that RQ is synthesized from UQ. Therefore, in parasitic helminths RQ might be synthesized by modification of UQ obtained from the host. However, we demonstrated that in F. hepatica adults RQ was not produced by modification of UQ obtained from the host but that RQ was synthesized de novo, as (i) the chain-length of the quinones of F. hepatica adults was not related to be chain length of the quinone of the host, (ii) despite many attempts we could never detect any in vitro conversion of UQ9 into RQ9 or into UQ10, neither by intact adult flukes nor by homogenates of F. hepatica adults and (iii) F. hepatica adults used mevalonate as precursor for the synthesis of RQ. We also showed that the rate of quinone synthesis in F. hepatica adults was comparable to that in the free-living nematode Caenorhabditis elegans. These results prompted the suggestion that RQ is synthesized via a pathway nearly identical to that of UQ biosynthesis: possibly only the last reaction differs